Troubleshooting for Labeling in vitro
Troubleshooting for Cellular Labeling
If solubility problems occur with your CLIP-tag fusion protein, we recommend testing a range of pH (pH 5.0–pH 10.0) and ionic strengths. The salt concentration may also need to be optimized for your particular fusion protein (50–250 mM).
Loss of Protein Due to Aggregation or Sticking to Tube
If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The CLIP-tag activity is not affected by this concentration of Tween 20.
If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Increase the incubation time to two hours total at 25°C or to 24 hours at 4°C; or halve the volume of protein solution labeled. Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the CLIP-tag using CLIP-Vista Green.
If the CLIP-tag fusion has been stored in the absence of DTT or other reducing agent, or has been stored at 4°C for a prolonged period, its activity may be compromised. Include 1 mM DTT in all solutions of the CLIP-tag fusion protein, and store the fusion protein at -20°C.
Using less than the recommended amount of substrate stock solution can significantly slow down the reaction rate.
Loss of Activity of Protein of Interest
If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the labeling time or decreasing the labeling temperature. If you label at 4°C we recommend overnight incubation.
If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is confirmed, check for expression of the CLIP-tag fusion protein via Western Blot using Anti-SNAP-tag Antibody (NEB #P9310). This antibody shows high crossreactivity with the CLIP-tag and can be used for Western Blot detection. Alternatively, CLIP-Vista Green (NEB #S9235) can be used to confirm presence of CLIP-tag fusion in cell extracts following SDS-PAGE, without the need for Western blotting.
Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CLIP-tag substrate and/or the incubation time. Improving the protein expression may also improve the signal. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling.
Legal and Disclaimers
Cellular Imaging and Analysis (i.e., SNAP and CLIP products)
The products and/or their use may be covered by one or more of the following patents and patent applications:
7,939,284 (Methods for Using O6-Alkylguanine-DNA-Alkyltransferases)
7,888,090 (Mutants of O6-Alkylguanine-DNA-Alkyltransferases)
8,163,479 Specific Substrates for O6-Alkylguanine-DNA-Alkyltransferases)
8,178,314 (Pyrimidines Reacting With O6-Alkylguanine-DNA-Alkyltransferases)
PCT/EP2007/057597 (Labeling of Fusion Proteins with Synthetic Probes)
EP07117800 (Drug Delivery)
EP07117802 (Drug Delivery)
EP07120288 (GTPase-Transient Protein Protein Interactions)
These patents and patent applications are owned by Covalys, or owned by the Ecole Polytechnique Fédérale de Lausanne (EPFL) and exclusively licensed to Covalys and NEB.